Enzymes are exquisite biocatalysts mediating every biological process in living organisms. In eukaryote cells, most enzymes are not freely diffused within the cytosols, but are spatially defined within subcellular organelles or closely co-localized as enzyme complexes along with other proteins (see, e.g. Conrado et al., Curr Opin Biotech 19, 492-499, (2008); Yan et al., J Proteomics 72, 4-11, (2009)). In consecutive reactions catalyzed by multiple enzymes, such close confinement minimizes diffusion of intermediates among the enzymes, enhancing overall reaction efficiency and specificity (see, e.g. Yan et al., J Proteomics 72, 4-11, (2009)).
Typically in vivo, toxic intermediates generated during a metabolic process are promptly eliminated by the proximate enzymes designed to degrade such toxins and which are co-localized within the confined structures (see, e.g. Kristensen et al., Natl Acad Sci USA 102, 1779-1784, (2005)). Peroxisomes, as an example, harbor a variety of oxidases with important metabolic and catabolic functions; toxic intermediates, such as hydrogen peroxide (H2O2) (see, e.g. Wanders et al., Annu Rev Biochem 75, 295-332, (2006); and Schrader et al., Histochem Cell Biol 122, 383-393, (2004)). Interestingly, nature circumvents this dilemma by incorporating catalase (Cat) within the peroxisomes. Catalase is highly active and specific in decomposing H2O2; rapid elimination of the as-generated H2O2 prevents its diffusion out from the peroxisomes and damage to other cellular components (see, e.g. Fujiwara et al., J Biol Chem 275, 37271-37277, (2000); and Sheikh et al., P Natl Acad Sci USA 95, 2961-2966, (1998)).
Although multiple-enzyme architectures commonly exist in nature, current enzyme-based applications, including enzyme catalysis and therapeutics, are still limited to the use of single enzyme or their mixtures (see, e.g. Leader et al., Nat Rev Drug Discov 7, 21-39, (2008); Iso et al., J Microencapsul 6, 165-176, (1989); Nouaimi-Bachmann et al., Biotechnol Bioeng 96, 623-630, (2007); and Mateo et al., Enzyme Microb Tech 40, 1451-1463, (2007)). Inorganic and polymeric materials incorporating multiple enzymes have been extensively studied; however, the enzymes were randomly immobilized within these materials in the forms of thin films or large particles (˜100 μm in diameter), excluding their therapeutic applications (see, e.g., Srere et al., P Natl Acad Sci USA 70, 2534-2538, (1973); Mansson et al., P Natl Acad Sci. Biol 80, 1487-1491, (1983); Kochschmidt et al., Eur J Biochem 81, 71-78, (1977); and Sheldon et al., Adv Synth Catal 349, 1289-1307, (2007)).
Fusion-protein techniques provide one strategy to construct multiple-enzyme architectures (see, e.g. Riedel et al., Mol Microbiol 28, 767-775, (1998); Shibuya et al., Biosci Biotech Bioch 56, 884-889, (1992); and Mao et al., J Biol Chem 270, 18295-18300, (1995). Ideally, such architectures with designable functions may be achieved by judiciously engineering the genomic inputs transfected to the host cells; nonetheless, this approach often leads to a loss or decreased enzyme specificity and activity (see, e.g. Stempfer et al., Nat Biotechnol 14, 481-484, (1996); and Seo et al., Appl Environ Microb 66, 2484-2490, (2000)). Moreover, finding suitable host cells and designing suitable spacers further place hurdles in the construction of fusion proteins (see, e.g. Bulow et al., Bio-Technol 3, 821-823, (1985)). Recently, post-translational assemblies were used to construct enzyme complexes; however, such an assembling process solely relies on specific bindings between the enzyme components and lacks general applicability (see, e.g., Mingardon et al., Appl Environ Microb 73, 7138-7149, (2007); Fierobe et al., J Biol Chem 276, 21257-21261, (2001); and Fierobe et al., J Biol Chem 277, 49621-49630, (2002)).